This invention was made with U.S. government support awarded to the following agency: NIH grants HL47250 and RR01008. The U.S. has certain rights in this invention.
Homocysteinemia is defined as the presence of the amino acid homocysteine in the blood. Although homocysteine is normally present in the blood of healthy humans at concentrations of 3-5 xcexcM, homocysteinemia generally refers to the medical condition of hyperhomocysteinemia, a situation in which plasma homocysteine concentrations are elevated (62, 84, 108). It has been proposed to classify homocysteinemia as moderate (15-30 xcexcM), intermediate (30-100 xcexcM), and severe ( greater than 100 xcexcM) on the basis of fasting plasma homocysteine concentrations (50).
Elevated levels of plasma homocysteine (homocysteinemia) are present in several pathologies (2, 6-7, 17, 30, 33, 35, 47, 50, 52-53, 59, 62-67, 68-70, 72-76, 81-84, 96, 105, 107-111, 114-116, 118, 121). The precise mechanisms by which homocysteinemia is related to the etiology of disease remains unknown. Inborn errors of amino acid metabolism in the transulfuration and remethylation pathways (In the metabolism of sulfur-containing amino acids, homocysteine is formed under physiological conditions by the demethylation of methionine. Homocysteine can either be remethylated by methylfolate homocysteine methyltransferase, or it may proceed to be converted to cystathionine and then to cysteine by the sequential actions of the enzymes cystathionine xcex2-synthase (CBS) and cystathionase, respectively). For many years, deficiencies of these enzymes have been recognized in children to cause homocysteine to be excreted in the urine (homocysteinuria). Children suffering from this rare genetic condition rarely survive into adulthood and develop atherosclerosis and thromboembolic disorders (9, 89). Recently, it has been shown that another genetic cause of homocysteinemia is a mutation of the methylfolate homocysteine methyltransferase gene (25-26, 83, 118). Certain of these individuals appear to be susceptible to the development of premature vascular disease. Despite the plethora of epidemiological evidence linking homocysteinemia to the development of atherosclerosis and thromboembolic disorders, the role of homocysteine in these conditions remains unclear.
The cyclic thioester, homocysteine thiolactone, has been implicated as a compound, which may be formed in conditions of homocysteinemia (equation 1) (63-67). Homocysteine thiolactone has been shown to react with primary amines by forming an amide linkage (equation 2) (1). Once formed in aqueous solution at physiological pH and temperature, homocysteine thiolactone is stable with a half-life of 25 hours (45). The half-life will decrease, however, in the presence of primary amines. For example, in the presence of excess cysteine, or lysine, the half-life is reported to be 3 hours (45). In addition, the reactivity of homocysteine thiolactone toward primary amines will increase at a higher pH (e.g. 8.0-8.4). However, it has been demonstrated that this molecule is unstable at high pH and will hydrolyze to form homocysteine (13). It has been reported that homocysteine thiolactone is capable of modifying LDL to a form which is capable of generating foam cells (72). While there is evidence that indicates homocysteine thiolactone may be involved in atherosclerosis, arteriosclerosis, and thromboembolic disorders, a method for the detection of the homocystamide adduct has been lacking.
Autoantibodies recognizing oxidized LDL, malondialdehyde-lysyl-LDL adduct, or 4-hydroxynoneal-lysyl-LDL adduct have been found to occur in vivo (8, 77, 120). In addition, derivitization of albumin, fibrinogen, or LDL lysyl residues by carbamylation, acetylation, ethylation, and methylation has been shown to result in high affinity antibodies directed against these modifications in experimental animals (99). These studies show that LDL may be a good carrier molecule for the immunization of animals (99). It has been suggested that analogous biochemistry between homocysteine thiolactone and LDL, resulting in homocystamide-LDL adducts, could result in the formation of autoantibodies against these adducts, although the notion of homocysteine thiolactone in vivo was considered physiologically irrelevant (equation 3, FIG. 1A) (14-15).
In one embodiment, the present invention is an antibody or a portion thereof which specifically forms a complex with a homocystamide adduct. The antibody may be either a monoclonal or polyclonal antibody and the homocystamide adduct is preferably selected from the group consisting of homocystamide-LDL, homocystamide-BSA and homocystamide-lysine.
Preferably, the affinity of the antibody for the homocystamide adduct is at least such that the IC50 is less than 15 xcexcM.
In another embodiment, the present invention is a method of measuring homocysteine levels in the sample comprising the steps of obtaining the sample, treating the sample such that endogenous homocysteine is converted to homocystamide adduct of either exogenous or endogenous carrier molecules, exposing the sample to the antibody described above, and correlating the binding of the antibody to a standard antibody binding profile. In this way, one may measure homocysteine levels in samples such as human plasma.
The present invention is also a method of evaluating endogenous homocystamide adducts comprising the steps of obtaining the sample, exposing the sample to the antibody of claim 1 and correlating the antibody binding to a standardized antibody binding profile.
It is an object of the present invention to measure homocysteine levels in both biological and non-biological samples.
It is another object of the present invention to diagnostically predict a patient""s susceptibility to vascular occlusive disorders.
It is another object of the present invention to evaluate the endogenous homocystamide adduct level in biological and non-biological samples.
It is another object of the present invention to provide an antibody specific for homocystamide adducts.
Other objects, advantages and features of the present invention will become apparent to one of skill in the art after one has evaluated the specification, claims and drawings.